Oligomerization and the sensitivity of phosphoenolpyruvate carboxylase to inactivation by proteinases

Phosphenolpyruvate (PEP) carboxylase from leaves of Crassula argentea displays varying levels of sensitivity to inactivation by various proteolytic enzymes. In general, the native enzyme is sensitive to proteinases known to attack at the carbonyl end of lysine or arginine (trypsin, papain, or bromelain). The ineffective proteolytic enzymes are those which have low specificity or which attack at the N-terminal end of hydrophobic amino acids, or which cannot attack lysine. The lack of an effect of endoproteinase arginine C, which is specific for arginine, probably indicates that lysine is the critical residue. When the native enzyme, which is comprised of an equilibrium of dimers with tetramers in approximately equal quantities, is treated by preincubation with 5 millimolar PEP, the enzyme becomes much more resistant to proteolytic inactivation. When the preincubation is with 5 millimolar malate rather than buffer alone, the effect is to slightly increase (ca. 15%) the sensitivity of the enzyme to inactivation by trypsin as measured by estimates of the pseudo-first order rate constant for inactivation. PEP carboxylase from corn leaves appears to be relatively susceptible to inactivation by trypsin, but is unaffected by preincubation with malate or PEP. The sensitivity of this C₄ enzyme to inhibition by malate is also unaffected by preincubation with these ligands.     

Development of the alt Mutant of Pisum sativum L

The alt (albina-terminalis) mutant of Pisum sativum L. germinates normally, produces several nodes, and then above a sharp transition produces 2 to 3 bleached nodes, ceases growth, and eventually dies. Green nodes have normal chlorophyll content, absorption spectra, photosynthetic rates, and ultrastructure. In bleaching tissues, the chloroplasts degenerate rapidly, followed by extensive disruption and loss of the remaining cytoplasm and organelles. Application of tissue extracts of normal genotypes of pea, corn, and bean stimulates apical development of alt. The resulting tissues have essentially normal structure and function. Application of thiamine, thiamine monophosphate, and thiamine pyrophosphate also stimulate normal apical development at concentrations of 1 micromolar and above. Partial characterization of the stimulus from pea seed extracts is consistent with thiamine as the active factor.     

Transport Properties of the Tomato Fruit Tonoplast : I. Identification and Characterization of an Anion-Sensitive H-ATPase

An anion-sensitive H⁺-translocating ATPase was identified in membrane vesicles isolated from mature green tomato (Lycopersicon esculentum) fruit. The H⁺-ATPase was associated with a low density membrane population having a peak density of 1.11 grams per cubic centimeter, and its activity was inhibited by NO₃⁻, N,N′-dicyclohexylcarbodiimide and diethylstilbestrol but not by vanadate, azide, molybdate, or oligomycin. This H⁺-ATPase has an unusual pH dependence indicating both a slightly acidic and a near neutral peak of activity. Chloride was found to be a potent stimulator of ATPase activity. The K_m for the H⁺-ATPase was approximately 0.8 millimolar ATP. The characteristics of this H⁺-ATPase are very similar to those described for a number of plant cell tonoplast H⁺-ATPases suggesting that the activity identified in tomato fruit membranes is tonoplast-associated. This report demonstrates the feasibility of isolating tonoplast vesicles from acidic fruit tissues for studies of transport activities associated with fruit development and maturation.     

Immunocytochemical Localization of Phosphoribulose Kinase in the Cyanelles of Cyanophora paradoxa and Glaucocystis nostochinearum

The distribution of phosphoribulose kinase (PRK) in the cyanelles of Cyanophora paradoxa Korschikoff and Glaucocystis nostochinearum Itzigsohn was studied by protein A-gold immunoelectron microscopy. In both endocyanomes, antiserum against PRK heavily labeled the thylakoid region of the cyanelles, whereas little or no label was present over the carboxysomes. Antiserum against ribulose 1,5-bisphosphate carboxylase/oxygenase by contrast heavily labeled the carboxysomes of each endocyanome. In vitro studies of PRK distribution in cell-free extracts of C. paradoxa showed that 93% of the enzyme was in the soluble fraction. Quantitative immunoelectron microscopy showed that more than 99% of the PRK in the cyanelle of C. paradoxa was localized in the thylakoid region. We conclude that the carboxysomes of cyanelles like the carboxysomes of autotrophic prokaryotes and the pyrenoids of green algal chloroplasts do not contain phosphoribulose kinase.     

Identification of polysaccharide hydrolases involved in autolytic degradation of zea cell walls

Cell walls of Zea mays (cv L.G.11) seedlings labeled with ¹⁴C were treated with α-amylase from Bacillus subtilis to remove starch and mixed linkage glucans. These walls released arabinose, xylose, galactose, and galacturonic acid in addition to glucose when they were allowed to autolyze. Methylation analysis was performed on samples of wall which had been incubated autolytically and the results indicated that degradation of the major polymer of the wall, the glucoarabinoxylan, had occurred. A number of glycanases could be dissociated from the wall by use of 3 m LiCL. The proteins which were released were found to contain a number of exoglycosidase activities in addition to being effective in degrading the polysaccharide substrates, araban, xylan, galactan, laminarin, mannan, and polygalacturonic acid. The effects of these enzymes on the wall during autolysis appear to result from endo-activity in addition to exo-activity. The structural changes that occurred in the cell walls during autolysis were found to be related to the changes previously found to occur in cell walls during auxin induced extension.     

Amino Acid Metabolism of Lemna minor L. : II. Responses to Chlorsulfuron

Chlorsulfuron, an inhibitor of acetolactate synthase (EC 4.1.3.18) (TB Ray 1984 Plant Physiol 75: 827-831), markedly inhibited the growth of Lemna minor at concentrations of 10⁻⁸ molar and above, but had no inhibitory effects on growth at 10⁻⁹ molar. At growth inhibitory concentrations, chlorsulfuron caused a pronounced increase in total free amino acid levels within 24 hours. Valine, leucine, and isoleucine, however, became smaller percentages of the total free amino acid pool as the concentration of chlorsulfuron was increased. At concentrations of chlorsulfuron of 10⁻⁸ molar and above, a new amino acid was accumulated in the free pool. This amino acid was identified as α-amino-n-butyrate by chemical ionization and electron impact gas chromatography-mass spectrometry. The amount of α-amino-n-butyrate increased from undetectable levels in untreated plants, to as high as 840 nanomoles per gram fresh weight (2.44% of the total free pool) in plants treated with 10⁻⁴ molar chlorsulfuron for 24 hours. The accumulation of this amino acid was completely inhibited by methionine sulfoximine. Chlorsulfuron did not inhibit the methionine sulfoximine induced accumulations of valine, leucine, and isoleucine, supporting the idea that the accumulation of the branched-chain amino acids in methionine sulfoximine treated plants is the result of protein turnover rather than enhanced synthesis. Protein turnover may be primarily responsible for the failure to achieve complete depletion of valine, leucine, and isoleucine even at concentrations of chlorsulfuron some 10⁴ times greater than that required to inhibit growth. Tracer studies with ¹⁵N demonstrate that chlorsulfuron inhibits the incorporation of ¹⁵N into valine, leucine, and isoleucine. The α-amino-n-butyrate accumulated in the presence of chlorsulfuron and [¹⁵N]H₄⁺ was heavily labeled with ¹⁵N at early time points and appeared to be derived by transamination from a rapidly labeled amino acid such as glutamate or alanine. We propose that chlorsulfuron inhibition of acetolactate synthase may lead to accumulation of 2-oxobutyrate in the isoleucine branch of the pathway, and transamination of 2-oxobutyrate to α-amino-n-butyrate by a constitutive transaminase utilizing either glutamate or alanine as α-amino-N donors.     

Effect of tagetitoxin on the levels of ribulose 1,5-bisphosphate carboxylase, ribosomes, and RNA in plastids of wheat leaves

Growth of wheat seedlings in the presence of the phytotoxin tagetitoxin produces pigment-deficient leaves of normal size and morphology whose cells contain only rudimentary plastids. We could not detect the accumulation of either the plastid-encoded large subunit or the nuclear-encoded small subunit of the chloroplast stromal enzyme ribulose 1,5-bisphosphate carboxylase (RuBPCase) in western blots of protein extracted from leaves of such seedlings. Sucrose gradient centrifugation profiles showed that plastid ribosomes were essentially absent in toxin-treated leaf tissue while cytoplasmic ribosomes were relatively unaffected. Northern blot analysis of RNA in toxin-treated leaves showed a deficiency of plastid ribosomal RNA (16S and 23S) as well as reduced levels of plastid mRNAs for the large subunit of RuBPCase and for the 32 kilodalton thylakoid Q_B polypeptide. Northern analysis also showed that the nuclear-encoded rbcS mRNA for the small subunit of RuBPCase is present in only trace amounts in toxin-treated leaves.     

Protein Synthesis in Bromegrass (Bromus inermis Leyss) Cultured Cells during the Induction of Frost Tolerance by Abscisic Acid or Low Temperature

Bromus inermis Leyss cell cultures treated with 75 micromolar abscisic acid (ABA) at both 23 and 3°C developed more freezing resistance than cells cultured at 3°C. Protein synthesis in cells induced to become freezing tolerant by ABA and low temperature was monitored by [¹⁴C]leucine incorporation. Protein synthesis continued at 3°C, but net cell growth was stopped. Most of the major proteins detected at 23°C were synthesized at 3°C. However, some proteins were synthesized only at low temperatures, whereas others were inhibited. ABA showed similar effects on protein synthesis at both 23 and 3°C. Comparative electrophoretic analysis of [¹⁴C]leucine labeled protein detected the synthesis of 19, 21 and 47 kilodalton proteins in less than 8 hours after exposure to exogenous ABA. Proteins in the 20 kilodalton range were also synthesized at 3°C. In addition, a 31 kilodalton protein band showed increased expression in freezing resistant ABA treated cultures after 36 hours growth at both 3 and 23°C. Quantitative analysis of [¹⁴C]leucine labeled polypeptides in two-dimensional gels confirmed the increased expression of the 31 kilodalton protein. Two-dimensional analysis also resolved a 72 kilodalton protein enriched in ABA treated cultures and identified three proteins (24.5, 47, and 48 kilodaltons) induced by low temperature growth.     

Partial Purification and Characterization of Uracil-DNA Glycosylase Activity from Chloroplasts of Zea mays Seedlings

A uracil-DNA glycosylase activity has been purified about 750-fold from the chloroplasts of light-grown Zea mays seedlings. This report represents the first direct demonstration of a DNA-glycosylase repair activity in chloroplasts. The activity, in part, was associated with a chloroplast Triton X-100 sensitive membrane. Its apparent K_m was 1.0 micromolar for a poly(dA-dT/U) substrate, and its molecular weight, as determined by gel filtration, was 18,000. The enzyme exhibited optimal activity at pH 7.0 with an atypically narrow pH tolerance. Activity was inhibited greater than 60% by 10 millimolar NaCl, 5 millimolar MgCl₂, or 5 millimolar EDTA. Enzyme activity was inhibited 80% by 10 millimolar N-ethylmaleimide, a sulfhydryl group-blocking agent. The activity removed uracil more rapidly from single-stranded DNA than from double-stranded DNA. With this report, uracil-DNA glycosylase activity has now been attributed to all three DNA-containing organelles of eucaryotic cells.     

Influence of chloroplast development on the activation of the diphenyl ether herbicide acifluorfen-methyl

The activity of acifluorfen-methyl (AFM); methyl 5-(2-chloro-4-[trifluoromethyl] phenoxy)-2-nitrobenzoate in excised cucumber cotyledons (Cucumis sativus L.) was examined. AFM induced membrane disruption, was significantly greater when etiolated cotyledons were illuminated 16 hours at 150 microeinsteins per square meter per second photosynthetically active radiation versus incubation under illumination of 4-fold greater intensity. These results were unexpected since the loss of membrane integrity is initiated by photodynamic reactions. Untreated, etiolated cotyledons were not able to accumulate chlorophyll under the higher light intensity while control and herbicide treated cotyledons greened significantly under the lower intensity illumination suggesting that some process associated with greening stimulated AFM activity. Inhibition of greening by cycloheximide also reduced AFM activity. Intermittent lighting induced greening in AFM treated cotyledons without causing any detectable loss of plasmalemma integrity. Utilization of this system for pretreatment of cotyledons prior to continuous illumination revealed that activity was greater when tissue was greened in the presence of AFM than when herbicide treatments were made after a greening period of the same duration. The results indicate that the pigments in situ in etiolated tissue are sufficient, without greening, to initiate membrane disruption by AFM. However, greening increases the herbicidal efficacy greatly. Furthermore, the stimulation appears to be due to specific interactions between AFM and the developing plastid and is not attributable solely to an increase in endogenous photosensitizers.